SRI International and a group of collaborators propose to further develop the EcoCyc database (DB). EcoCyc is a freely and openly available model-organism DB for the bacterium Escherichia coli K-12 that is accessible to scientists through the World Wide Web, and may be downloaded for local installation. EcoCyc is heavily used by scientists from multiple disciplines. It serves as a general reference source on E. coli for experimental biologists, and is particularly useful for the analysis of functional-genomics experiments. The DB serves computational biologists who undertake global studies of E. coli, metabolic engineers developing new methods for chemicals production including biofuels researchers, and bioinformaticists who use EcoCyc as a gold-standard dataset to develop new computational methods. The DB is also used by educators. We propose to continue work to update EcoCyc in an ongoing fashion to reflect new information about E. coli genes, metabolic pathways, and regulatory interactions. Information will be fused from the biomedical literature and from high-throughput experiments. New types of data to be integrated include data on gene essentiality, on conditions of growth and non-growth for E. coli, experimental evidence for protein existence, protein localization information, and enzyme kinetics data. We will also undertake a comprehensive and ongoing effort to computationally validate the metabolic network model within EcoCyc by validating its predictions against many conditions of growth and non-growth for wildtype and knock-out strains of E. coli. We will develop bioinformatics methods for hypothesizing changes to the EcoCyc metabolic model that brings its predictions into closer concordance with large amounts of experimental data. The project will also expand the Pathway Tools software used to query and analyze EcoCyc, such as by modernizing elements of its graphical interface, adding sequence-related operations, and enabling retrieval of omics datasets from the EcoliHub resource. The project will develop educational materials based on EcoCyc for graduate and undergraduate education.